This invention relates to the fields of molecular biology and genetic transformation in higher plants. More specifically, the invention relates to novel uses of genes and their encoded proteins that participate in a disease resistance pathway(s) in multicellular plants.
Several publications are referenced in this application in parentheses in order to more fully describe the state of the art to which this invention pertains. Full citations for these references are found at the end of the specification. The disclosure of each of these publications is incorporated by reference herein.
Plant disease resistance is frequently associated with the formation of necrotic lesions, known as the hypersensitive response (HR), alterations in cell wall structure at the sites of infection, increases in endogenous salicylic acid (SA) levels, and activation of a complex array of defense-related genes, including the pathogenesis-related (PR) genes. In addition to these local responses, the uninfected portions of the plant usually develop systemic acquired resistance (SAR), which is manifested as enhanced resistance to a subsequent challenge by the initial or even unrelated pathogens. Activation of these defense responses is usually governed by a xe2x80x98gene-for-genexe2x80x99 interaction between a plant resistance (R) gene and a pathogen avirulence (Avr) gene, or initiated by the plant recognition of non-race-specific elicitors such as elicitins.
Plant recognition of pathogens occurs either at the surface of plasma membrane or in the cytoplasm. Recent studies have revealed that various components in the plant defense signaling pathway(s) exhibit structural and functional conservation to those identified in animals. For example, several R gene products, including the N gene (which confers resistance to tobacco mosaic virus (TMV) in tobacco) share homology with the interleukin-1 receptor and Toll protein, both of which are involved in the induction of immune responses in mammals and Drosophila, respectively. In addition, a variety of signaling events, such as Ca2+ flux, H2O2 burst generated by the activation of an NADPH oxidase, protein phosphorylation/dephosphorylation, and generation of oxylipin signaling molecules, have been associated with the induction of plant and animal defense responses.
Protein kinases and phosphatases have been implicated, through the use of their inhibitors, in the induction of several defense responses including medium alkalization, reactive oxygen species generation, defense gene activation and hypersensitive cell death. Kinase activities with characteristics of protein kinase C or MAP kinase have been associated with these processes. The MAP kinase cascade is one of the major pathways by which extracellular stimuli are transduced into intracellular responses in yeast and mammalian cells. In mammals, two of the three subfamilies of the MAP kinase family, the stress-activated protein kinase/Jun N-terminal kinase (SAPK/JNK) and the p38 kinase, are activated in response to various stress signals, including UV and ionizing radiation, hyperosmolarity, oxidative stress and cytokines.
A variety of MAP kinase genes have been isolated by PCR-based homology cloning from several plant species (Hirt, 1997; Mizoguchi et al., 1997). In addition, several kinase activities believed to be MAP kinases, based on the tact that they preferentially phosphorylate myelin basic protein (MBP) and are themselves phosphorylated on tyrosine residues upon activation, have been shown to be activated by stress stimuli. These include the tobacco wounding (cutting)-activated 46-kD kinase (Seo et al., 1995; Usami et al., 1995), the fungal elicitor-activated 47-kD kinase from tobacco (Suzuki and Shinshi, 1995), the harpin-activated 49-kD kinase from tobacco (xc3x81dxc3xa1m et al., 1997), and the wounding-, systemin- and oligosaccharide-activated 48-kD kinase from tomato (Stratmann and Ryan, 1997).
Studies using an antibody against the C-terminal peptide of the alfalfa MMK4 have linked the alfalfa MMK4 to cold, drought and mechanical stresses (Jonak et al., 1996; Bxc3x6gre et al., 1997). The same antibody was also used to demonstrate that parsley ERMK may encode the 45-kD MBP kinase activated by Pep25 elicitor derived from the Phytophthora sojae glycoprotein elicitor (Ligterink et al., 1997).
A 48-kD SA-induced protein kinase, termed SIPK, was identified in tobacco and its corresponding gene has been cloned using peptide sequences obtained by microsequencing of the purified protein (Zhang and Klessig, 1997; see also co-pending U.S. patent application Ser. No. 08/837,593, now U.S. Pat. No. 5,977,442, incorporated by reference herein). This MAP kinase was recently shown to be activated by various fungal elicitors (Zhang et al., 1998) and also by wounding (Zhang and Klessig, 1998) and by tobacco mosaic virus (TMV) infection (U.S. Ser. No. 08/837,593, now U.S. Pat. No. 5,977,442).
The aforementioned wounding-activated 46-kD protein kinase heretofore was believed to be encoded by WIPK, a member of tobacco MAP kinase family, since this gene is rapidly induced at the mRNA level by wounding (Seo et al., 1995). However, a rigorous demonstration of this has been lacking.
Genes that encode components of signal transduction pathways which are used by a plant to activate defense responses for protection against disease-causing agents can be used in a variety of ways to improve or enhance the disease resistance response in plants. Accordingly, a need exists to identify new genes that participate in such functions or, alternatively, to determine if certain genes that are already available also possess such functions.
In accordance with the present invention, it has now been discovered that the MAP kinase encoded by the tobacco WIPK gene is not activated by, and therefore not involved in, response to wounding, but rather is activatable in association with development or enhancement of resistance to microbial pathogens. Accordingly, the WIPK gene and its functional homologs in other species, and their encoded gene products, are useful for a variety of purposes relating to improving and enhancing a plant""s disease resistance.
According to one aspect of the invention, a transgenic plant is provided, which exhibits enhanced resistance to plant disease-causing agents such as viruses (such as TMV), fungi (such as Phytophthora spp.), bacteria (such as Pseudomonas spp.) and nematodes. The transgenic plant is stably transformed with a DNA construct, expressible in the cell, encoding a WIPK enzyme. The WIPK coding sequence from tobacco is preferred for use in the DNA construct.
According to another aspect of the invention, a method of making a transgenic plant with enhanced disease resistance is provided. The method comprises (1) transforming regenerable cells of a plant with a recombinant DNA construct, expressible in a plant, encoding a WIPK enzyme; and (2) regenerating a transgenic plant from those transformed cells. For reasons described in greater detail below, such plants are expected to exhibit enhanced resistance to a variety of disease-causing agents, including viruses, bacteria, fungi and nematodes.
The novel functions of WIPK identified in accordance with this invention and described in greater detail below, indicate that WIPK may play a key role in signal transduction for activation of plant defenses against microbial pathogens or components thereof. Accordingly, the new methods, plant cells and plants of the invention offer a significant advance in the field of plant molecular biology, as it pertains to enhancing the plant disease resistance response.